U.S. patent application number 11/569040 was filed with the patent office on 2008-09-11 for method for the detection of disease-related prion.
This patent application is currently assigned to PRIONICS AG. Invention is credited to Karin Biffiger, Franziska Kuhn, Bruno Oesch, Mario Purro, Alex Raber, Jaqueline Schmid, Daniel Zwald.
Application Number | 20080220447 11/569040 |
Document ID | / |
Family ID | 34924999 |
Filed Date | 2008-09-11 |
United States Patent
Application |
20080220447 |
Kind Code |
A1 |
Purro; Mario ; et
al. |
September 11, 2008 |
Method for the Detection of Disease-Related Prion
Abstract
Methods for the detection of the disease associated conformation
of the prion protein as an indication of transmissible spongiform
encephalopathies (TSEs), including preclinical detection of
infected live animals and humans, and post-mortem detection methods
are disclosed. In one aspect, the tissue or body fluid sample of a
test subject is contacted with an antibody that binds only the
disease related conformation of the prion protein under
non-denaturing conditions.
Inventors: |
Purro; Mario; (Zurich,
CH) ; Zwald; Daniel; (Wettingen, CH) ; Schmid;
Jaqueline; (Sellenburen, CH) ; Biffiger; Karin;
(Kirchdorf, CH) ; Kuhn; Franziska; (Zurich,
CH) ; Oesch; Bruno; (Stilli, CH) ; Raber;
Alex; (Zurich, CH) |
Correspondence
Address: |
RANKIN, HILL & CLARK LLP
38210 Glenn Avenue
WILLOUGHBY
OH
44094-7808
US
|
Assignee: |
PRIONICS AG
Schlieren
CH
|
Family ID: |
34924999 |
Appl. No.: |
11/569040 |
Filed: |
May 13, 2005 |
PCT Filed: |
May 13, 2005 |
PCT NO: |
PCT/EP05/05279 |
371 Date: |
November 13, 2006 |
Current U.S.
Class: |
435/7.8 ;
436/501 |
Current CPC
Class: |
G01N 33/6896 20130101;
G01N 2800/2828 20130101 |
Class at
Publication: |
435/7.8 ;
436/501 |
International
Class: |
G01N 33/53 20060101
G01N033/53; G01N 33/566 20060101 G01N033/566 |
Foreign Application Data
Date |
Code |
Application Number |
May 14, 2004 |
EP |
04011470.4 |
Claims
1. A method for the detection of an isoform (PrPD) of the prion
protein in body fluids and fractions of body fluids, especially
blood or urine, wherein the isoform PrPD is related to TSE and is
present as either protease sensitive isoform or as protease
resistant isoform, the method comprising the steps of: a. treatment
of the body fluid or its fraction with a capture antibody capable
of recognizing a conformational epitope on the TSE-related isoform
PrPD, said epitope being specific for the TSE-related isoform PrPD
but not necessarily indicative for the protease resistant isoform
of the prion protein; and b. detecting possible PrPD-capture
antibody complexes.
2. The method according to claim 1, wherein the body fluid includes
PrPD only as protease sensitive isoform.
3. The method according to claim 1, wherein the body fluid or its
fraction is treated with an additional detection antibody capable
of recognizing the N-terminus of prion-protein (PrP).
4. A method according to claim 1, wherein the method to detect PrPD
is carried out on plasma prepared from a blood sample.
5. The method according to claim 4, wherein the plasma sample is
treated with chemicals to allow optimal binding conditions for the
antibody/PrPD complex.
6. The method according to claim 1, wherein the antibody 15B3 is
used as capture antibody.
7. The method according to claim 1, wherein the anti-body/PrPD
complex is detected directly or indirectly by an immunological
assay.
8. Use of a method according to claim 1, for testing blood or blood
fractions, especially in connection with transfusion of human
blood.
9. The method according to claim 2, wherein the body fluid or its
fraction is treated with an additional detection antibody capable
of recognizing the N-terminus of prion-protein (PrP).
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to methods for the
detection of prion diseases in animals and humans.
BACKGROUND OF THE INVENTION
[0002] Transmissible spongiform encephalopathies (TSEs) or prion
diseases are fatal neurodegenerative diseases in animals and man.
The onset of clinical illness is preceded by a long incubation
period of months to decades. Clinical symptoms of TSEs include
dementia and loss of movement coordination. In the 1980s it was
found that a common hallmark of TSEs was the accumulation of an
abnormal protease resistant isoform (PrP.sup.res or PrP.sup.Sc) of
the host-encoded prion protein (PrP.sup.C) in affected animals and
humans. The discovery of PrP.sup.Sc provided a molecular marker
that was shown to be specific for all prion diseases as well as the
major and very likely the only, constituent of the infectious
particle, denominated prion.
[0003] The protease resistant core of PrP.sup.Sc, designated
PrP27-30, was discovered by analyzing proteinase K-treated
fractions with an apparent molecular mass of 27-30 kDa from Syrian
hamster brain for scrapie infectivity. N-terminal sequencing of
PrP-27-30 led to the cloning of the host-encoded PrP gene and the
identification of the protease-sensitive isoform of the prion
protein (PrP.sup.C) in uninfected animals. The key pathogenic event
in TSEs is the conformational change of the host-encoded prion
protein (PrP.sup.C) into the pathological isoform named PrP.sup.Sc
(after its first identification in experimentally scrapie-infected
rodents). This conformational change involves refolding of
.alpha.-helical structures of PrP.sup.C into .beta.-sheet of
PrP.sup.Sc. This refolded prion protein, PrP.sup.Sc, differs from
PrP.sup.C only in its tertiary structure and, thus, has the same
amino acid sequence, as well as the same post-translational
modifications such as the N-linked glycosylation and the
GPI-anchor. PrP.sup.Sc aggregates into amyloid fibrils and
accumulates in nervous tissue and to a lesser extent in
lymphoreticular tissues.
[0004] In an infected host, levels of PrP.sup.Sc are directly
proportional to prion titers. After experimental inoculation of
rodents with TSE agents, PrP.sup.Sc is usually detectable in the
central nervous system weeks before the appearance of disease, and
its level increases to a maximum that is reached up to months
before the animal dies. During the asymptomatic phase, both
infectivity and PrP.sup.Sc are readily detected in lymphoreticular
tissues. Recently, PrP.sup.Sc has also been found to accumulate in
muscle tissue of hamsters orally infected with scrapie and in
experimentally infected transgenic mice.
[0005] While the prototype of all prion diseases, scrapie in sheep
and goats, has been known for more than two centuries, a new form
of animal prion disease designated bovine spongiform encephalopathy
(BSE) has since its first recognition in the UK in 1986 developed
into a zoonosis. To what extent BSE infected cattle have entered
the human food chain is still a matter of debate and has been the
subject of a number of studies. A recent study published by the
Imperial College in London (Donnelly, C A., Ferguson, N.M., Ghani,
A. C. & Anderson, R. M., Statistical Methods in Medical
Research 12, 177-190 (2003)) suggested that based on new
epidemiological data up to 1.6 Mio BSE infected cattle could have
ended up on the plate of British consumers.
[0006] The human prion diseases comprise Creutzfeldt-Jakob disease
(CJD), Gerstmann-Straeussler-Scheinker disease (GSS), fatal
familial insomnia (FFI) and kuru. These diseases illustrate the
three manifestations of prion diseases in general, namely the
sporadic forms of the disease (80-90% of all CJD cases), the
inherited forms linked to mutations in the human PrP gene (familial
GSS, familial CJD and FFI) and the infectious forms which are
acquired by transplantation, injection or ingestion of
prion-contaminated tissue-derived products (iatrogenic CJD, vCJD
and kuru). With the emergence of a new form of Creutzfeldt-Jakob
disease in the UK in 1996, a new episode in the battle against
human diseases caused by food-borne pathogens has been initiated.
Up to date, 146 cases of variant CJD (vCJD) have been reported in
the UK, and compelling scientific evidence argues for a causal
relationship between BSE and vCJD. With no obvious risk factors
identified in these patients, a causal link to the exposure of
BSE-contaminated food products seems very likely. Detecting vCJD
before patients show clinical symptoms is an urgent priority, as it
could dramatically reduce the risk of contaminating blood supplies
and hospital equipment. Recently, a patient in the UK developed
vCJD 6.5 years after receiving a blood transfusion from a donor who
developed symptoms of vCJD 3.5 years after the blood donation
(Llewelyn et al., 2004, The Lancet 363, 417-421). This raised the
possibility that vCJD might be transmitted by blood transfusion and
underscores the need for a diagnostic test to detect prions in
blood and fractions thereof. In the present invention a method is
disclosed to detect the disease-associated form of PrP in plasma of
TSE infected mammals.
[0007] Because PrP.sup.Sc is the only reliable molecular marker for
prion diseases, immunological detection in brain tissue of the
protease resistant part of PrP.sup.Sc is the basis for rapid
diagnostic tests that are currently used for active surveillance of
BSE and scrapie. Due to the slow kinetics of accumulation of
PrP.sup.Sc in the preclinical stage of the disease, the current
diagnostic capabilities are strongly limited with respect to the
detection of the disease early in the incubation period. The
invention disclosed herein provides methods for the detection of
prion diseases in the preclinical stage.
[0008] While PrP.sup.Sc was originally defined as a partially
protease resistant and detergent insoluble isoform of PrP, several
prion diseases have been identified which are associated with
abnormal PrP lacking the classical protease resistance. Forms of
PrP that are associated with these prion diseases are termed
`protease sensitive` PrP.sup.Sc to distinguish them from the
protease resistant forms of PrP.sup.Sc.
[0009] The detection of the disease related conformation of the
prion protein in tissues of an animal or human is thought to be
diagnostic of prion disease. To distinguish the disease related PrP
from its cellular precursor PrP.sup.C, either treatment with
proteases to completely destroy PrP.sup.C, but only remove a
protease-sensitive N-terminal part of PrP.sup.Sc, is required, or
alternatively an antibody that is able to distinguish between
PrP.sup.C and PrP.sup.Sc can be used. One such antibody that has
been shown to react only with native PrP.sup.Sc but not with
PrP.sup.C has been disclosed in our patent application WO 98/37210
and in Korth et al. (1997, Nature 390: 74-77) entitled
`Immunological detection of prions` which is incorporated herein by
reference to disclose and describe such antibodies.
[0010] Most tests that are currently used for the diagnosis of
prion diseases are carried out on tissue of the central nervous
system which is obtained post mortem from an animal or human either
showing clinical disease or suspected of having the disease. The
diagnostic tests rely on the use of a pre-treatment to hydrolyse
the protease sensitive form of the prion protein followed by the
detection of the protease resistant form of the prion protein by an
immunological assay. However, such assays will not detect the
protease sensitive form of PrP.sup.Sc. For example, the patent
application WO 00/52197 claims a method to detect PrP.sup.Sc in
serum of a TSE infected mammal. A major drawback of this method is
the use of a pre-treatment with proteases to hydrolyse protease
sensitive PrP.
[0011] There is an urgent need to provide simple and robust methods
for the detection of protease resistant and protease sensitive
forms of PrP that are associated with TSEs and diagnostic kits
allowing detection of the disease in live mammals.
BRIEF SUMMARY OF THE INVENTION
[0012] It is an object of the present invention to overcome the
drawbacks and failures of prior art and to provide methods and
diagnostic kits for the detection of prion diseases in live animals
and humans.
[0013] Such a method would offer the advantage to detect carriers
of a transmissible spongiform encephalopathy by screening blood for
the presence of disease related PrP and could e.g. prevent vCJD
from being accidentally transmitted by blood transfusion.
DEFINITIONS
[0014] The term `PrP` refers to the common amino acid sequence of
the prion protein isoforms rather than to the different
conformations.
[0015] The term `PrP.sup.C` refers to the prion protein isoform
that is abundant in normal mammals and is not related to TSE.
[0016] The term `PrP.sup.D` refers to a disease (TSE) related form
of PrP which is defined as a conformationally altered isoform of
the normal host prion protein. PrP.sup.D includes protease
resistant and protease sensitive forms of disease associated PrP.
The term PrP.sup.D is used synonymously: for conformationally
altered e.g. ovine, bovine or human PrP, to cite only some
examples.
[0017] The term `antibody` refers to any antibody generated by
immunization of an animal or by in vitro selection/panning
procedures. The antibody may be polyclonal or monoclonal, including
fragments thereof such as Fab or single chain antibodies. The
antibody may be genetically engineered, such as chimeric or
humanized.
[0018] According to the present invention a method is disclosed to
detect PrP.sup.D in an animal or human including taking a sample,
preferably blood or other body fluids which are easily obtained
from a live mammal, then the sample is brought into contact with an
antibody recognizing only PrP.sup.D, and finally the amount of
antibody/PrP.sup.D complex is detected by using an immunological
assay.
[0019] The method of the invention takes into account that the
isoform PrP.sup.D related to TSE can be present either as protease
sensitive isoform or as protease resistant isoform. In this context
it is provided that the sample is treated with a special capture
antibody capable of recognizing a conformational epitope on the
TSE-related isoform PrP.sup.D. The epitope is chosen to be specific
for the TSE-related isoform PrP.sup.D, but not necessarily
indicative for the protease resistant isoform of the prion protein.
By using a capture antibody recognizing the described
conformational epitope, one obtains optimal results especially in
samples which may contain the TSE-related PrP.sup.D alternatively
in either the protease sensitive or protease resistant isoform.
[0020] In a preferred embodiment, the method is used to analyse
samples which include PrP.sup.D only in the protease sensitive
isoform. Such samples can be processed only by using a capture
antibody according to the invention.
[0021] In a preferred embodiment of the invention, the method to
detect PrP.sup.D is carried out on plasma prepared from a blood
sample according to a procedure that is part of this invention.
[0022] In another preferred embodiment, the plasma sample is
treated with chemicals to allow optimal binding conditions for the
antibody/PrP.sup.D complex.
[0023] In another preferred embodiment of the invention, the
antibody 15B3 is used to detect only PrP.sup.D, but not the normal
form of PrP. The antibody 15B3 is available from Prionics AG,
Zurich, Switzerland and methods to generate such antibodies have
been disclosed in WO 98/37210. Hybridomacells capable of producing
antibody 15B3 have been deposited in connection with WO 98/37210 at
DSMZ--Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
under Accession number: DSM ACC2298.
[0024] In yet another preferred embodiment of the invention, the
antibody/PrP.sup.D complex is detected directly or indirectly by an
immunological assay.
[0025] Another preferred embodiment of the invention provides a
method for the detection of the antibody/PrP.sup.D complex using a
sandwich ELISA with a labelled antibody that recognizes the
N-terminus of the prion protein.
[0026] A preferred embodiment of the invention is to use the method
for the detection of PrP.sup.D for testing human blood samples to
decide whether the blood is safe for transfusion.
[0027] In another preferred embodiment of the invention diagnostic
kits are provided for the diagnosis of TSEs in live mammals.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 is a graph showing the results of Example 1. In this
example a sandwich immunoassay was performed using 15B3 as a
capture antibody and an antibody recognizing the N-terminus of PrP
as a detection antibody on plasma samples from BSE infected (Pos
1-Pos 2) versus normal cows (Neg 1-Neg 23).
[0029] FIG. 2 is a graph showing the results of Example 2. In this
example a sandwich immunoassay was performed using 15B3 as a
capture antibody and an antibody recognizing the N-terminus of PrP
as a detection antibody on plasma samples from scrapie infected
(Pos1-Pos6) versus normal sheep (Neg 1-Neg 16).
[0030] FIG. 3 is a graph showing the results of Example 3. The data
shown illustrate that scrapie positive brain homogenate (B) can be
discriminated from Scrapie negative brain homogenate (A) using FACS
analysis. The grey curve shows the fluorescence signal received
with beads coated with antibody 15B3. The black line shows the
fluorescence signal obtained with isotype control beads.
[0031] FIG. 4 is a graph showing the results of Example 4. The data
shown illustrate that Serum originating from a scrapie infected
sheep (B) can be discriminated from a Serum sample originating from
a scrapie uninfected sheep (A) using FACS analysis. The right upper
quadrant set at 5% in the negative sample (A) was assumed in the
positive sample (B) and clearly shows a shift from 5 to 32%.
[0032] FIG. 5 is a graph showing the results of Example 5. The data
shown illustrate that white blood cells originating from a scrapie
infected sheep (B) can be discriminated from a white blood cells
sample originating from a scrapie uninfected sheep (A) using FACS
analysis. The right upper quadrant set at 5% in the negative sample
(A) was assumed in the positive sample (B) and clearly shows a
shift from 5 to 36.9%.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
[0033] According to the invention, the method for detection of
PrP.sup.D in the diagnosis of TSEs comprises:
1) collecting a blood sample from an animal or a human 2) preparing
total blood, plasma, serum, red blood cells (RBCs) or white blood
cells (WBCs) from blood sample and treatment of plasma for the
binding of the analyte PrP.sup.D with the antibody 3) incubating
the blood sample with a PrP.sup.D specific antibody as capture
antibody 4) washing the antibody/PrP.sup.D complex to remove
contaminating proteins 5) detecting the antibody/PrP.sup.D complex
with a sensitive immunological assay.
[0034] In the following, the individual steps will be described in
more detail with reference to the specific examples. It is to be
understood that the disclosed method and components are not limited
to particular samples, chemical agents, antibodies, labels, or
assays as such may vary. Unless defined otherwise, all technical
and scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs.
[0035] A blood sample is withdrawn from a mammal and placed in a
container that contains an anticoagulant such as EDTA, sodium
citrate or heparin. A preferred anticoagulant is EDTA but others
may be used with similar results. Separation of plasma from the
blood cells is performed by centrifugation at 1000.times.g. Other
preferred samples are body fluids such as saliva, urine or
cerebrospinal fluid. If serum is used, the blood is withdrawn into
a container with no anticoagulants and the blood is allowed to clot
for at least 30 minutes at room temperature followed by
centrifugation at 1000.times.g to separate the blood clot from
serum.
[0036] The plasma sample is mixed with an equal volume of buffer
containing a detergent to prevent the unspecific binding of
PrP.sup.c to the antibody. Vast varieties of detergents that are
suitable for blocking unspecific binding exist and are known to the
person skilled in the art. Preferred detergents are, sodium
laurylsarcosine, sodium deoxy cholate, sulfobetaine, Triton X-100,
NP-40, ZWITTERGENT, Tween-20, Tween-80. The preferred detergent
used in a particular assay format and for a particular species may
vary. Preferred concentrations are between 0.05 and 1%.
[0037] The plasma sample is brought into contact with the antibody.
In a preferred embodiment of the invention an antibody that is
specific for the disease associated form PrP.sup.D is used. A
preferred antibody is the antibody 15B3 which has been shown to be
specific for PrPD but other antibodies may be used in a similar
fashion. In a particularly preferred embodiment the antibody is
coated on the wells of a microtitre plate or to beads to capture
PrPD, in the sample. The complex of the antibody with PrP.sup.D is
then detected in an immunological assay using a detection antibody
that recognizes PrP. Detection can be achieved by any immunological
method. Such assays are well known to those skilled in the art and
may include Western blotting (immunoblotting), enzyme linked
immunosorbent assays (ELISA), immuno-PCR or fluorescence activated
cell sorting (FACS). Direct detection may be carried out by using
an antibody to the prion protein that is conjugated with an enzyme
such as peroxidase, alkaline phosphatase or fluorescent molecules
or nucleic acids. Alternatively, the detection can be indirectly
using an unlabelled primary (anti prion protein antibody) and a
labelled secondary antibody.
[0038] The invention is illustrated in the following non-exclusive
examples and accompanying figures:
[0039] EXAMPLE 1: Determination of PrP.sup.D in plasma from BSE
negative and positive cattle.
[0040] EXAMPLE 2: Determination of PrP.sup.D in plasma from scrapie
negative and positive sheep.
[0041] EXAMPLE 3: Determination of PrP.sup.D in brain homogenate
from scrapie negative and positive sheep.
[0042] EXAMPLE 4: Determination of PrP.sup.D in serum from scrapie
negative and positive sheep.
[0043] EXAMPLE 5: Determination of PrP.sup.D in white blood cells
from scrapie negative and positive sheep.
Example 1
[0044] The graph in FIG. 1 shows the results of a first experiment.
The data shown illustrate that BSE positive plasma samples can be
distinguished from negative samples using the sandwich immunoassay.
The mean of double measurements are shown, whereas the error bars
indicate the difference to the higher value.
[0045] 96-well plates were coated with 2 .mu.g/ml anti-IgM antibody
diluted in phosphate buffered saline (PBS) pH 7.4, 0.1% bovine
serum albumin (BSA) for 1 hour at room temperature. Unbound
antibody was removed by washing the plates three times with PBS pH
7.4 containing 0.05% Tween-20. Thereafter, the plates were blocked
for 2 hours and washed three times with PBS pH 7.4 containing 0.05%
Tween-20. Conformation specific antibody 15B3 in PBS pH 7.4 at a
concentration of 2 .mu.g/ml was bound to anti-IgM for 1 hour at
room temperature. After washing the plate three times with PBS pH
7.4 containing 0.05% Tween 20, 110 .mu.l Plasma from BSE negative
and positive cattle diluted 1:1 with Tris buffered saline (TBS)
containing 0.4% deoxycholate (DOC) was added to the plate and
incubated for 1.5 hours at room temperature. Unbound proteins were
washed away with TBS containing 0.2% DOC. Peroxidase labelled
monoclonal antibody 805 recognizing amino acids 25-40 of PrP was
diluted to 10 ng/ml in a buffer containing PBS pH 7.4, 0.02%
Casein, 0.1% Tween-20 and added for 1 hour to wells. Unbound
antibody was removed by washing for three times with PBS pH 7.4
containing 0.05% Tween-20. 100 ml of chemiluminescent substrate
solution was added to the wells and read in a plate luminometer.
The values are given as relative light units.
Example 2
[0046] The graph in FIG. 2 shows the results of a second
experiment. The data shown illustrate that Sheep scrapie positive
plasma samples can be distinguished from negative samples using the
sandwich immunoassay. The mean of double measurements are shown
whereas the error bars indicate the difference to the higher
value.
[0047] Coating of plate with 15B3 antibody was performed as
described in example 1. 110 .mu.l of Plasma from scrapie negative
and positive sheep diluted 1:1 with TBS containing 0.2% Sarcosyl
was added to the plate and incubated for 1.5 hours at room
temperature. Unbound proteins were washed away with TBS containing
0.1% Sarcosyl. POD labelled monoclonal antibody 805 recognizing
amino acids 25-40 of PrP was diluted to 10 ng/ml in a buffer
containing PBS pH 7.4, 0.02% Casein, 0.1% Tween-20 and added for 1
hour to wells. Unbound antibody was removed by washing for three
times with PBS pH 7.4 containing 0.05% Tween-20. 100 .mu.l of
chemiluminescent substrate solution was added to the wells and read
in a plate luminometer. The values are given as relative light
units.
Example 3
[0048] The graph in FIG. 3 shows the results of a third experiment.
The data shown illustrate that scrapie positive brain homogenate
can be distinguished from negative samples using FACS analysis. A
histogram is shown whereas y-axis indicates the events and x-axis
the relative intensity of fluorescence measured by the FL-2
detector.
[0049] 60 ng monoclonal 15B3 antibody or Isotype control IgM
antibody (Becton Dickinson 550963) were coated on 3 .mu.l magnetic
anti-IgM Dynabeads (Dynal 110.15) in phosphate buffer saline (PBS)
pH 7.4 containing 0.1% bovine serum albumin (BSA) for 1 hour at
room temperature on a rotating wheel. Unbound antibody was removed
by washing the beads three times with PBS pH 7.4 containing 0.1%
BSA. The beads were added to 1 ml Tris buffered saline (TBS)
containing 0.25% Sarkosyl and 10 .mu.l of a 10% scrapie positive or
negative brain homogenate and incubated over night at 4.degree. C.
on a rotating wheel. Thereafter, unbound proteins were removed by
washing the beads three times with TBS containing 0.2% Sarkosyl.
The beads were thereafter added to 200 .mu.l of PBS pH 7.4, 0.1%
Tween-20, 0.02% Casein containing 2 .mu.g of biotinylated
monoclonal antibody 805 and incubated for 15 min at room
temperature on a rotating wheel. After washing for three times with
200 .mu.l PBS pH 7.4 containing 0.1% Tween-20 and 0.02% Casein 2.5
.mu.g of Streptavidine-Phycoerythrin conjugate (Becton Dickinson
554061) in 200 .mu.l PBS pH 7.4, 0.1% Tween-20, 0.02% Casein was
added and incubated for an additional 5 min at room temperature.
After removing unbound conjugate by washing beads were taken up in
400 .mu.l PBS pH 7.4, 0.1% Tween-20, 0.02% Casein and analysed by
FACS (Fluorescence Activated Cell Sorting) (MoFlow). Acquisition
was performed at linear mode for FSC/SSC and log mode for FL2. Data
acquisition was performed in list mode. FL2 base-line was set on
15B3 beads without prior sample incubation.
Example 4
[0050] The graph in FIG. 4 shows the results of a fourth
experiment. The data shown illustrate that scrapie positive serum
(B) can be distinguished from negative samples (A) using FACS
analysis. A dot blot is shown whereas the y-axis indicate the size
of the particles (Forward scatter) and the x-axis the relative
intensity of fluorescence measured by the FL2 detector.
[0051] 60 ng monoclonal 15B3 antibody were coated on 3 .mu.l
magnetic anti-IgM Dyna-beads (Dynal 110.15) in phosphate buffer
saline (PBS) pH 7.4 containing 0.1% bovine serum albumin (BSA) for
1 hour at room temperature on a rotating wheel. Unbound antibody
was removed by washing the beads three times with PBS pH 7.4
containing 0.1% BSA. The beads were added to 500 .mu.l Tris
buffered saline (TBS) containing 0.2% Sarkosyl and 250 .mu.l of a
serum originating from a scrapie infected respectively scrapie
uninfected animal and incubated over night at 4.degree. C. on a
rotating wheel. Thereafter unbound proteins were removed by washing
the beads three times with TBS containing 0.2% Sarkosyl. The beads
were thereafter added to 200 .mu.l of PBS pH 7.4, 0.1% Tween-20,
0.02% Casein containing 2 .mu.g of biotinylated monoclonal antibody
805 and incubated for 15 min at room temperature on a rotating
wheel. After washing for three times with 200 .mu.l PBS pH 7.4
containing 0.1% Tween-20 and 0.02% Casein 2.5 .mu.g of
Streptavidine-Phycoerythrin conjugate (Becton Dickinson 554061) in
200 .mu.l PBS pH 7.4, 0.1% Tween-20, 0.02% Casein was added to the
beads and incubated for an additional 5 min at room temperature.
After washing unbound conjugate away, beads were taken up in 400
.mu.l PBS pH 7.4, 0.1% Tween-20, 0.02% Casein and analyzed by FACS
(MoFlow). Acquisition was performed at linear mode for FSC/SSC and
log mode for FL2. Data acquisition was performed in list mode. FL2
base-line was set on 15B3 beads without prior sample
incubation.
Example 5
[0052] The graph in FIG. 5 shows the results of a fifth experiment.
The data shown illustrate that white blood cells extracted from a
scrapie positive (B) sheep can be distinguished from scrapie
negative ones (A) using FACS analysis. A dot blot is shown whereas
the y-axis indicate the size of the particles (Forward scatter) and
the x-axis, the relative intensity of fluorescence measured by the
FL2 detector.
[0053] 60 ng monoclonal 15B3 antibody were coated on 3 .mu.l
magnetic anti-IgM Dyna-beads (Dynal 110.15) in phosphate buffer
saline (PBS) pH 7.4 containing 0.1% bovine serum albumin (BSA) for
1 hour at room temperature on a rotating wheel. Unbound antibody
was removed by washing the beads three times with PBS pH 7.4
containing 0.1% BSA. The beads were added to 500 .mu.l Tris
buffered saline (TBS) containing 0.2% Sarkosyl and 2 millions white
blood cells originating from a scrapie infected respectively
scrapie uninfected animal and incubated over night at 4.degree. C.
on a rotating wheel. Thereafter, unbound proteins were removed by
washing the beads three times with TBS containing 0.2% Sarkosyl.
The beads were thereafter added to 200 .mu.l of PBS pH 7.4, 0.1%
Tween-20, 0.02% Casein containing 2 .mu.g of biotinylated
monoclonal antibody 805 and incubated for 15 min at room
temperature on a rotating wheel. After washing for three times with
200 .mu.l PBS pH 7.4 containing 0.1% Tween-20 and 0.02% Casein 2.5
.mu.g of Streptavidine-Phycoerythrin conjugate (Becton Dickinson
554061) in 200 .mu.l PBS pH 7.4, 0.1% Tween-20, 0.02% Casein was
added to the beads and incubated for an additional 5 min at room
temperature. After washing unbound conjugate away beads were taken
up in 400 .mu.l PBS pH 7.4, 0.1% Tween-20, 0.02% Casein and
analyzed by FACS (MoFlow). Acquisition was performed at linear mode
for FSC/SSC and log mode for FL2. Data acquisition was performed in
listmode. FL2 base-line was set on 15B3 beads without prior sample
incubation.
* * * * *